The expression of Catsup in the hindgut is essential for zinc homeostasis in Drosophila melanogaster.

Zinc excretion is crucial for zinc homeostasis. However, the mechanism of zinc excretion has not been well characterized. Zinc homeostasis in Drosophila seems well conserved to mammals. In this study, we screened all members of the zinc transporters ZnT (SLC30) and Zip (SLC39) for their potential roles in Drosophila hindgut, an insect organ that belongs to the excretory system. The results indicated that Catecholamines up (Catsup, CG10449), a ZIP member localized to the Golgi, is responsible for zinc homeostasis in the hindgut of Drosophila hindgut-specific knockdown of Catsup leads to a developmental arrest in the larval stage, which could be rescued well by human ZIP7. Further study suggested that Catsup RNAi in the hindgut reduced zinc levels in the excretory system (containing the Malpighian tubule and hindgut) but exhibited systemic zinc overload. Besides, more calculi were observed in the Malpighian tubules of Catsup RNAi flies. The developmental arrest and calculi in the Malpighian tubules of hindgut-specific Catsup RNAi flies could be rescued by dietary zinc restriction but hypersensitivity to zinc. These results will help us understand the fundamental process of zinc excretion in higher eukaryotes.

Fear-of-intimacy-mediated zinc transport is required for Drosophila fat body endoreplication.

BACKGROUND: Endoreplication is involved in the development and function of many organs, the pathologic process of several diseases. However, the metabolic underpinnings and regulation of endoreplication have yet to be well clarified. RESULTS: Here, we showed that a zinc transporter fear-of-intimacy (foi) is necessary for Drosophila fat body endoreplication. foi knockdown in the fat body led to fat body cell nuclei failure to attain standard size, decreased fat body size and pupal lethality. These phenotypes could be modulated by either altered expression of genes involved in zinc metabolism or intervention of dietary zinc levels. Further studies indicated that the intracellular depletion of zinc caused by foi knockdown results in oxidative stress, which activates the ROS-JNK signaling pathway, and then inhibits the expression of Myc, which is required for tissue endoreplication and larval growth in Drosophila. CONCLUSIONS: Our results indicated that FOI is critical in coordinating fat body endoreplication and larval growth in Drosophila. Our study provides a novel insight into the relationship between zinc and endoreplication in insects and may provide a reference for relevant mammalian studies.

The expression of Catsup in escort cells affects Drosophila ovarian stem cell niche establishment and germline stem cells self-renewal via Notch signaling.

Stem cell niche provides extrinsic signals to maintain stem cell renewal or initiate cell differentiation. Drosophila niche is composed of somatic terminal filament cells, cap cells and escort cells. However, the underlying mechanism for the development of stem cell niche remains largely unclear. Here we found that the expression of a zinc transporter Catsup is essential for ovary morphogenesis. Catsup knockdown in escort cells results in defects of niche establishment and germline stem cells self-renewal. These defects could be modified by altered expression of genes involved in zinc metabolism or intervention of dietary zinc levels. Further studies indicated that Catsup RNAi affected adult ovary morphogenesis by suppressing Notch signaling. Lastly, we demonstrated that the defects of Catsup RNAi could be restored by overexpression of heat shock cognate protein 70 (Hsc70). These findings expand our understanding of the mechanisms controlling adult oogenesis and niche establishment in Drosophila.

Drosophila ZIP13 over-expression or transferrin1 RNAi influences the muscle degeneration of Pink1 RNAi by elevating iron levels in mitochondria.

Disruption of iron homeostasis in the brain of Parkinson's disease (PD) patients has been reported for many years, but the underlying mechanisms remain unclear. To investigate iron metabolism genes related to PTEN-induced kinase 1 (Pink1) and parkin (E3 ubiquitin ligase), two PD-associated proteins that function to coordinate mitochondrial turnover via induction of selective mitophagy, we conducted a genetic screen in Drosophila and found that altered expression of genes involved in iron metabolism, such as Drosophila ZIP13 (dZIP13) or transferrin1 (Tsf1), significantly influences the disease progression related to Pink1 but not parkin. Several phenotypes of Pink1 mutant and Pink1 RNAi but not parkin mutant were significantly rescued by over-expression (OE) of dZIP13 (dZIP13 OE) or silencing of Tsf1 (Tsf1 RNAi) in the flight muscles. The rescue effects of dZIP13 OE or Tsf1 RNAi were not exerted through mitochondrial disruption or mitophagy; instead, the iron levels in mitochondira were significantly increased, resulting in enhanced activities of enzymes participating in respiration and increased ATP synthesis. Consistently, the rescue effects of dZIP13 OE or Tsf1 RNAi on Pink1 RNAi can be inhibited by decreasing the iron levels in mitochondria through mitoferrin (dmfrn) RNAi. This study suggests that dZIP13, Tsf1, and dmfrn might act independently of parkin in a parallel pathway downstream of Pink1 by modulating respiration and indicates that manipulation of iron levels in mitochondria may provide a novel therapeutic strategy for PD associated with Pink1.

Zinc antagonizes iron-regulation of tyrosine hydroxylase activity and dopamine production in Drosophila melanogaster.

BACKGROUND: Dopamine (DA) is a neurotransmitter that plays roles in movement, cognition, attention, and reward responses, and deficient DA signaling is associated with the progression of a number of neurological diseases, such as Parkinson's disease. Due to its critical functions, DA expression levels in the brain are tightly controlled, with one important and rate-limiting step in its biosynthetic pathway being catalyzed by tyrosine hydroxylase (TH), an enzyme that uses iron ion (Fe2+) as a cofactor. A role for metal ions has additionally been associated with the etiology of Parkinson's disease. However, the way dopamine synthesis is regulated in vivo or whether regulation of metal ion levels is a component of DA synthesis is not fully understood. Here, we analyze the role of Catsup, the Drosophila ortholog of the mammalian zinc transporter SLC39A7 (ZIP7), in regulating dopamine levels. RESULTS: We found that Catsup is a functional zinc transporter that regulates intracellular zinc distribution between the ER/Golgi and the cytosol. Loss-of-function of Catsup leads to increased DA levels, and we showed that the increased dopamine production is due to a reduction in zinc levels in the cytosol. Zinc ion (Zn2+) negatively regulates dopamine synthesis through direct inhibition of TH activity, by antagonizing Fe2+ binding to TH, thus rendering the enzyme ineffective or non-functional. CONCLUSIONS: Our findings uncovered a previously unknown mechanism underlying the control of cellular dopamine expression, with normal levels of dopamine synthesis being maintained through a balance between Fe2+ and Zn2+ ions. The findings also provide support for metal modulation as a possible therapeutic strategy in the treatment of Parkinson's disease and other dopamine-related diseases.

EGCG ameliorates neuronal and behavioral defects by remodeling gut microbiota and TotM expression in Drosophila models of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Eigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is known to exert a beneficial effect on PD patients. Although some mechanisms were suggested to underlie this intervention, it remains unknown if the EGCG-mediated protection was achieved by remodeling gut microbiota. In the present study, 0.1 mM or 0.5 mM EGCG was administered to the Drosophila melanogaster with PINK1 (PTEN induced putative kinase 1) mutations, a prototype PD model, and their behavioral performances, as well as neuronal/mitochondrial morphology (only for 0.5 mM EGCG treatment) were determined. According to the results, the mutant PINK1B9 flies exhibited dopaminergic, survival, and behavioral deficits, which were rescued by EGCG supplementation. Meanwhile, EGCG resulted in profound changes in gut microbial compositions in PINK1B9 flies, restoring the abundance of a set of bacteria. Notably, EGCG protection was blunted when gut microbiota was disrupted by antibiotics. We further isolated four bacterial strains from fly guts and the supplementation of individual Lactobacillus plantarum or Acetobacter pomorum strain exacerbated the neuronal and behavioral dysfunction of PD flies, which could not be rescued by EGCG. Transcriptomic analysis identified TotM as the central gene responding to EGCG or microbial manipulations. Genetic ablation of TotM blocked the recovery activity of EGCG, suggesting that EGCG-mediated protection warrants TotM. Apart from familial form, EGCG was also potent in improving sporadic PD symptoms induced by rotenone treatment, wherein gut microbiota shared regulatory roles. Together, our results suggest the relevance of the gut microbiota-TotM pathway in EGCG-mediated neuroprotection, providing insight into indirect mechanisms underlying nutritional intervention of Parkinson's disease.

Transferrin1 modulates rotenone-induced Parkinson's disease through affecting iron homeostasis in Drosophila melanogaster.

Mitochondrial dysfunction and oxidative stress are pathophysiologic mechanisms implicated in Parkinson's disease (PD). In recent years, environmental toxins are employed to increase oxidative stress mediated neuropathology and sporadic PD. Disruption of iron homeostasis has been implicated in PD patients for many years, but the functional role of iron in sporadic PD pathogenesis is still not well clarified in vivo. To address this question, we set out to investigate the effect of iron on a Drosophila rotenone model of sporadic PD. Iron homeostasis is maintained by many transporters. We found that inhibition of transferrin1 (Tsf1) expression in the central nervous system (CNS) results in reduced iron levels in brains and significantly ameliorates the neurodegenerative phenotypes of rotenone exposure Drosophila; moreover, the rotenone induced reactive oxygen species (ROS) levels in the brain, the damaged complex I activity and the decreased ATP generation were dramatically rescued by Tsf1 knockdown. Further study indicated that all the rescue effects of Tsf1 knockdown on sporadic PD could be inhibited by malvolio (Mvl) overexpression, an iron transporter responsible for iron uptake. These results imply that Tsf1 knockdown in the CNS could attenuate rotenone toxicity by decreasing the ROS levels in brains through reducing iron levels, and manipulation of iron transporters in brains may provide a novel therapeutic strategy for sporadic PD.

What can flies tell us about zinc homeostasis?

Zinc is an essential micronutrient for all organisms. For multicellular organisms, zinc uptake, storage, distribution and export are tightly regulated at both cellular and organismal levels, to cope with the multiple requirements versus the toxicity of the metal ion. During the past decade, the fruit fly Drosophila melanogaster has become an important model organism for the elucidation of metazoan zinc homeostasis. This review describes our current knowledge of various zinc transporters in Drosophila, with an emphasis on the process of dietary zinc uptake in the fly. We also discuss how Drosophila was used as a model to facilitate our understanding of the role of zinc in neurodegenerative diseases.

Huntington disease arises from a combinatory toxicity of polyglutamine and copper binding.

Huntington disease (HD) is a progressive neurodegenerative disorder caused by dominant polyglutamine (polyQ) expansion within the N terminus of huntingtin (Htt) protein. Abnormal metal accumulation in the striatum of HD patients has been reported for many years, but a causative relationship has not yet been established. Furthermore, if metal is indeed involved in HD, the underlying mechanism needs to be explored. Here using a Drosophila model of HD, wherein Htt exon1 with expanded polyQ (Htt exon1-polyQ) is introduced, we show that altered expression of genes involved in copper metabolism significantly modulates the HD progression. Intervention of dietary copper levels also modifies HD phenotypes in the fly. Copper reduction to a large extent decreases the level of oligomerized and aggregated Htt. Strikingly, substitution of two potential copper-binding residues of Htt, Met8 and His82, completely dissociates the copper-intensifying toxicity of Htt exon1-polyQ. Our results therefore indicate HD entails two levels of toxicity: the copper-facilitated protein aggregation as conferred by a direct copper binding in the exon1 and the copper-independent polyQ toxicity. The existence of these two parallel pathways converging into Htt toxicity also suggests that an ideal HD therapy would be a multipronged approach that takes both these actions into consideration.

Genetic inhibition of solute-linked carrier 39 family transporter 1 ameliorates aß pathology in a Drosophila model of Alzheimer's disease.

The aggregation or oligomerization of amyloid-ß (Aß) peptide is thought to be the primary causative event in the pathogenesis of Alzheimer's disease (AD). Considerable in vitro evidence indicates that the aggregation/oligomerization of Aß is promoted in the presence of Zn; however, the functional role of Zn in AD pathogenesis is still not well clarified in vivo. Zn is imported into the brain mainly through the solute-linked carrier (Slc) 39 family transporters. Using a genetically tractable Drosophila model, we found that the expression of dZip1, the orthologue of human Slc39 family transporter hZip1 in Drosophila, was altered in the brains of Aß42-expressing flies, and Zn homeostasis could be modulated by forcible dZip1 expression changes. An array of phenotypes associated with Aß expression could be modified by altering dZip1 expression. Importantly, Aß42 fibril deposits as well as its SDS-soluble form were dramatically reduced upon dZip1 inhibition, resulting in less neurodegeneration, significantly improved cognitive performance, and prolonged lifespan of the Aß42-transgenic flies. These findings suggest that zinc contributes significantly to the Aß pathology, and manipulation of zinc transporters in AD brains may provide a novel therapeutic strategy.

Angiotensin-converting enzyme inhibitory peptide IVGFPAYGH protects against liver injury in mice fed a high­sodium diet by inhibiting the RAS and remodeling gut microbial communities.

Consuming a high­sodium diet carries serious health risks and significantly influences the activation state of the renin-angiotensin system (RAS). This study evaluates the protective effect of angiotensin-converting enzyme (ACE) inhibitory peptide IVGFPAYGH on a high­sodium diet-induced liver injury. IVGFPAYGH supplementation increased the activities of liver antioxidase and decreased the levels of liver inflammatory factor in mice fed a high­sodium diet (8 % NaCl). IVGFPAYGH supplementation also reduced liver fatty acid synthesis and promoted fatty acid oxidation, increased the expression of low-density lipoprotein receptor, and improved liver dyslipidemia. Furthermore, IVGFPAYGH supplementation inhibited the activation of the liver RAS via inhibiting ACE activity and reducing angiotensin II levels in mice fed a high­sodium diet. Moreover, IVGFPAYGH supplementation could alter the gut microbiota composition toward a normal gut microbiota composition and increase the abundance of the Lactobacillus genus. IVGFPAYGH supplementation also increased the expression levels of small intestinal tight junction protein and cecum short-chain fatty acids. Thus, IVGFPAYGH supplementation may maintain intestinal homeostasis and improve high­sodium diet-induced liver injury by altering the gut microbiota composition and inhibiting the RAS. IVGFPAYGH is a promising functional ingredient for protecting liver damage caused by a high­sodium diet.

Partial Replacement of NaCl with KCl in Cooked Meat Could Reduce the Liver Damage Through Renin-Angiotensin System in Mice.

SCOPE: Dietary salt (sodium chloride, NaCl) is necessary for processed meat products, but intake of a high-sodium diet carries serious health risks. Considerable studies indicate that the partial substitution of NaCl with potassium chloride (KCl) can produce sodium-reduced cooked meat. However, most studies of sodium-reduced cooked meat focus on the production process in vitro, and the effect of cooked meat on health has not been well clarified in vivo. METHODS AND RESULTS: This study finds that compared to the high-sodium group (HS), serum renin, angiotensin-converting enzyme (ACE), angiotensin (Ang) II, and the levels of some indicators of dyslipidemia are decreased in the reduced salt by partial substitution of NaCl with KCl group (RS + K). Furthermore, RS + K increases the antioxidation abilities, inhibits the renin-angiotensin system (RAS) through ACE/Ang II/Ang II type 1 receptor axis pathway, reduces synthesis of triglyceride and cholesterol and protein expressions of inflammatory factors interleukin-17A and nuclear factor-kappa B in the liver. CONCLUSION: Partial substitution of NaCl with KCl in cooked meat can be a feasible approach for improving the health benefits and developing novel functional meat products for nutritional health interventions.

Assessing metal ion transporting activity of ZIPs: Intracellular zinc and iron detection.

Zrt/Irt-like proteins (ZIPs or SLC39A) are a large family of metal ion transporters mainly responsible for zinc uptake. Some ZIPs have been shown to specifically transport zinc, whereas others have broader substrate specificity in divalent metal ion trafficking, notably those of zinc and iron ions. Measuring intracellular zinc and iron levels helps assess their molecular and physiological activities. This chapter presents step-by-step methods for evaluating intracellular metal ion concentrations, including direct measurement using inductively coupled plasma-mass spectrometry (ICP-MS), chemical staining, fluorescent probes, and indirect reporter assays such as activity analysis of enzymes whose activities are dependent on metal ion availability.

Angiotensin I-Converting Enzyme Inhibitory Activity of Two Peptides Derived from In Vitro Digestion Products of Pork Sausage with Partial Substitution of NaCl by KCl.

This study aimed to identify angiotensin I-converting enzyme (ACE) from in vitro digestion products of pork sausage with partial substitution of NaCl by KCl (PSRK). Peptides from in vitro digestion products of PSRK were identified through liquid chromatography with tandem mass spectrometry analysis coupled with de novo sequencing. Subsequently, the ACE inhibitory peptides LIVGFPAYGH and IVGFPAYGH were screened based on PeptideRanker, in silico absorption, molecular docking, and the determination of ACE inhibitory activity. In addition, the ACE inhibitory peptides LIVGFPAYGH and IVGFPAYGH were mixed-type inhibitors; these peptides' ACE inhibitory activities were expressed as the 50% inhibitory concentration (IC50) values in vitro, which were 196.16 and 150.88 µM, respectively. After 2 h of incubation, LIVGFPAYGH and IVGFPAYGH could be transported through Caco-2 cell monolayers with paracellular passive diffusion. Furthermore, LIVGFPAYGH and IVGFPAYGH significantly increased the levels of ACE2 and nitric oxide while decreasing the levels of ACE, angiotensin II, and endothelin-1 in Ang I-treated human umbilical vein endothelial cells, indicating the ACE inhibitory effect of LIVGFPAYGH and IVGFPAYGH. In summary, LIVGFPAYGH and IVGFPAYGH from PSRK can be used as functional foods with antihypertensive activity.

In vitro digestibility of proteins, peptidomic analysis and antioxidant ability of sodium-reduced pork sausage with partial substitution of NaCl by KCl.

High salt (NaCl) consumption can impact on human health, and KCl is the most widely used replacement salt in meat products. This study investigated the effects of 0% NaCl (NS), 3% NaCl (HS), 1.95% NaCl (RS), 1.95% NaCl+1.05% KCl (RS + K) on protein digestibility of pork sausage in vitro. The results indicated that RS + K showed the highest gastrointestinal digestibility (GID) because of the structure of looser cross-linked strands and uniform cavities, while HS exhibited the lowest GID. RS + K released more peptides (2499) during gastrointestinal than NS (2301), RS (2130) and HS (2235), with a higher proportion of peptides with molecular weights <1000 Da, and more unique peptides. Meanwhile, the digestion product of RS + K exhibited excellent radical scavenging activity and improved the antioxidant abilities to reduce oxidative injury which was induced by H2O2 in HepG2 cells. These results demonstrated that partial substitution with KCl can be an effective strategy for improving the digestibility of sodium-reduced gel-type meat products.

Identification of antioxidant peptides targeting Keap1-Nrf2-ARE pathway from in vitro digestion of pork sausage with partial substitution of NaCl by KCl.

The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response elements (ARE) pathway is one of the most important cellular defense mechanisms against oxidative stress. This study focuses on finding antioxidant peptides from in vitro digestion products of pork sausage with partial substitution of NaCl by KCl by virtual screening. Six antioxidant peptides, LIVGFPAYGH, DWWGSTVR, WNSLLIR, IVGFPAYGH, FDNLWDQGL, and LRSPSWDPF, could activate the Keap1-Nrf2-ARE pathway and protect cells from oxidative stress. DWWGSTVR exhibits the most robust activity among them. Further studies indicated that DWWGSTVR could increase the expression of many antioxidant enzymes by enabling the transfer of Nrf2 from the cytoplasm to the nucleus. In summary, these six peptides are proven to be Nrf2 activators and could be used as functional foods to prevent and treat various oxidative stress-induced diseases.

Molecular physiology of zinc in Drosophila melanogaster.

New research in Drosophila melanogaster has revealed the molecular mechanisms of zinc involvement in many biological processes. A newly discovered Metallothionein is predicted to have a higher zinc specificity than the other isoforms. Zinc negatively regulates tyrosine hydroxylase activity by antagonizing iron binding, thus rendering the enzyme ineffective or non-functional. The identification of a new chaperone of the protein disulfide isomerase family provided mechanistic insight into the protein trafficking defects caused by zinc dyshomeostasis in the secretory pathway. Insect models of tumor pathogenesis indicate that zinc regulates the structural stabilization of cells by transcriptionally regulating matrix metalloproteinases while zinc dyshomeostasis in the secretory pathway modulates cell signaling through endoplastic recticulum stress.

Chicken skin-derived collagen peptides chelated zinc promotes zinc absorption and represses tumor growth and invasion in vivo by suppressing autophagy.

To improve the utilization value of chicken by-products, we utilized the method of step-by-step hydrolysis with bromelain and flavourzyme to prepare low molecular weight chicken skin collagen peptides (CCP) (<5 kDa) and characterized the amino acids composition of the CCP. Then, we prepared novel CCP-chelated zinc (CCP-Zn) by chelating the CCP with ZnSO4. We found that the bioavailability of CCP-Zn is higher than ZnSO4. Besides, CCP, ZnSO4, or CCP-Zn effectively repressed the tumor growth, invasion, and migration in a Drosophila malignant tumor model. Moreover, the anti-tumor activity of CCP-Zn is higher than CCP or ZnSO4. Furthermore, the functional mechanism studies indicated that CCP, ZnSO4, or CCP-Zn inhibits tumor progression by reducing the autonomous and non-autonomous autophagy in tumor cells and the microenvironment. Therefore, this research provides in vivo evidence for utilizing chicken skin in the development of zinc supplements and cancer treatment in the future.

Ursolic Acid Protects Sodium Dodecyl Sulfate-Induced Drosophila Ulcerative Colitis Model by Inhibiting the JNK Signaling.

Ursolic acid (UA) is a bioactive molecule widely distributed in various fruits and vegetables, which was reported to play a therapeutic role in ulcerative colitis (UC) induced by toxic chemicals. However, the underlying mechanism has not been well clarified in vivo. Here, using a Drosophila UC model induced by sodium dodecyl sulfate (SDS), we investigated the defensive effect of UA on intestinal damage. The results showed that UA could significantly protect Drosophila from the damage caused by SDS exposure. Further, UA alleviated the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by SDS and upregulated the activities of total superoxide dismutase (T-SOD) and catalase (CAT). Moreover, the proliferation and differentiation of intestine stem cells (ISCs) as well as the excessive activation of the c-Jun N-terminal kinase (JNK)-dependent JAK/STAT signaling pathway induced by SDS were restored by UA. In conclusion, UA prevents intestine injury from toxic compounds by reducing the JNK/JAK/STAT signaling pathway. UA may provide a theoretical basis for functional food or natural medicine development.

Methods to Assay the Behavior of Drosophila melanogaster for Toxicity Study.

Drosophila melanogaster, the fruit fly, has been widely used in biological investigation as an invertebrate alternative to mammals for its various advantages compared to other model organisms, which include short life cycle, easy handling, high prolificacy, and great availability of substantial genetic information. The behavior of Drosophila melanogaster is closely related to its growth, which can reflect the physiological conditions of Drosophila. We have optimized simple and robust behavioral assays for determining the larvae survival, adult climbing ability (mobility assay), reproductive behavior, and lifespan of Drosophila. In this chapter, we present the step-by-step detailed method for studying Drosophila behavior.